Genetic and phenotypic analysis in horses : parasitological and body growth parameters

Orientador : Prof. Dr. Marcelo Beltrão MolentoTese (doutorado) - Universidade Federal do Paraná, Setor de Ciências Agrárias, Programa de Pós-Graduação em Ciências Veterinárias. Defesa : Curitiba, 21/02/2018Inclui referênciasResumo: Ao longo das últimas décadas, os ciatostomíneos (pequenos estrôngilos) tornaram-se os parasitas internos mais importantes em cavalos. Sendo agentes de graves infecções parasitárias e com o controle cada vez mais difícil devido à seleção de populações resistentes à maioria dos grupos de anti-helmínticos disponíveis. Portanto, são necessários estudos para avaliar as infecções parasitárias e a influência dessas doenças no desenvolvimento corporal dos animais; assim como, descobrir os padrões genéticos que podem estar relacionados à sua infeção, apresentando alta ou baixa contagem de ovos por grama de fezes (OPG). O objetivo deste estudo foi realizar análises genéticas e fenotípicas de parâmetros parasitológicos e de desenvolvimento corporal em cavalos de puro sangue inglês (PSI) naturalmente parasitados. A presente tese está dividida em cinco capítulos, apresentados em formato de Introdução, manuscritos e as Considerações Finais. O primeiro artigo comparou duas técnicas de contagem de OPG, McMaster (McM) e Mini-FLOTAC (mF), para a detecção de ovos de nematódeos gastrointestinais de bovinos e equinos, em diferentes locais. Em todos os experimentos em média os valores do desvio padrão e do coeficiente de variação foram significativamente menores para a técnica de mF. Portanto, recomenda-se o uso da técnica MiniFLOTAC, que é um método com menor variabilidade e maior precisão, especialmente para animais com baixo OPG. O segundo artigo teve como objetivo avaliar o impacto do gênero do animal, mês de nascimento, idade e número de filhos da égua, e contagem de OPG na taxa de crescimento de cavalos PSI. Este estudo também teve como objetivo correlacionar as informações sobre o desempenho dos animais nas corridas associadas aos valores OPG. O peso corporal médio dos animais ao nascer foi 56,5 kg (machos) e 51,9 kg (fêmeas), e aos 18 meses, pesavam em média 435,9 e 441,6 kg, respectivamente. Altura da cernelha média ao nascer foi de 104,36 cm (machos) e 101,19 cm (fêmeas), e aos 18 meses, a altura foi de 154,78 e 153,5 cm, respectivamente. Os dados das corridas mostraram que os animais com maior OPG apresentaram um desempenho menor quando comparados com aqueles com menor OPG, incluindo um número menor de vitórias. Concluindo que animais criados sob um conjunto bem definido de práticas de manejo podem ter poucos ou nenhum sinal de comprometimento devido as infecções parasitárias. O objetivo do artigo 3 foi determinar regiões genômicas associadas aos padrões individuais do OPG dos animais, realizando um estudo de associação genômica ampla entre equinos PSI naturalmente infectados com parasitas gastrointestinais. Neste estudo, regiões candidatas relacionadas ao desenvolvimento e ativação do sistema imune foram encontradas nos cromosomos 17, 21 e 25. Esta tese destacou várias possibilidades para investigar parasitarias gastrointestinais em cavalos, melhorando a compreensão e o controle de infecções por ciatostomíneos. Observou-se que os cavalos criados sob um conjunto bem definido de práticas de manejo podem ter poucos ou nenhum sinal de comprometimento devido a infecções parasitárias. Palavras-chave: Equino. Ciatostomíneos. Cavalo. Desenvolvimento corporal. Mini-FLOTAC. OPG. GWAS.Abstract: Over the last decades, cyathostomins (small strongyles) have become the most important internal parasites of horses. They are agents of serious parasitic infections and their control is increasingly difficult due to the selection of resistant populations to most of the anthelmintic groups available. Therefore, studies to assess the parasite infections, and the influence of these diseases on body development of animals are necessary. In the same line, it is important to discover the genetic patterns of the animals that may be related to their infection, presenting high or low fecal egg count (FEC). The aim of this study was to perform genetic and phenotypic analysis of parasitological and body development parameters in Thoroughbred horses. The present Thesis is divided in five Chapters, presented in the format of Introduction, manuscripts and Final Considerations. The first article compared two FEC techniques, namely McMaster (McM) and Mini-FLOTAC (mF), for the detection of cattle and horse gastrointestinal nematode eggs, in different locations. On average, in all experiments, the standard deviation and the coefficient of variation values were significantly lower for mF. Therefore, it is recommended the use of the Mini-FLOTAC technique, which is a method with less variability and higher accuracy, particularly for animals with low FEC. The second article had the objective to evaluate the impact of animal gender, month of birth, age of mare, number of offspring and FEC in the growth rate of Thoroughbred horses. This study also aimed to correlate the information on racing careers associated with the FEC values of the animals. The mean body weight at birth was 56.5 kg for males and 51.9 kg for females, and at 18 months’, males and females weighed on average 435.9 and 441.6 kg, respectively. The mean withers height at birth was 104.36 cm for males and 101.19 cm for females, and at 18 months, the height was 154.78 and 153.5 cm, respectively. The racing history data showed that animals with higher FEC had a poorer performance when compared to those with lower FEC, including a smaller number of victories. In conclusion, it was suggested that horses raised under a welldefined set of management practices can have little or no signs of impairment due to worm infections. The aim of the article three was to determine genomic regions associated with Strongyle FEC patterns, conducting a genomic wide association study (GWAS) among Thoroughbred equines naturally infected with gastrointestinal parasites. In this study, candidate regions related to immune system development and activation were found in ECA17, ECA 21 and ECA25. This thesis has highlighted several possibilities to further investigate the control of gastrointestinal parasite infections in horses, improving the understanding and control of cyathostomins infections. It was observed that horses raised under a well-defined set of management practices can have little or no signs of impairment due to worm infections. Key-words: Equine. Horse. Cyathostomins. Body growth. Mini-FLOTAC. EPG. GWAS

Origanum vulgare (Lamiaceae) OVICIDAL POTENTIAL ON GASTROINTESTINAL NEMATODES OF CATTLE

Due to anthelmintic resistance in nematodes, several research studies have been developed seeking control alternatives to these parasites. This study evaluated the in vitro action of Origanum vulgare on gastrointestinal nematode eggs of cattle. In order to evaluate the ability to inhibit egg hatch, different dried leaves extracts of this plant were tested, such as dye, hydroalcoholic and aqueous extracts at concentrations varying from 0.62 to 80 mg/mL. Each assay was accompanied by control containing levamisole hydrochloride (0.2 mg/mL), distilled water and 70 ºGL grain alcohol at the same concentration of the extracts. Test results showed that the different O. vulgare extracts inhibited egg hatch of cattle gastrointestinal nematodes at a percentage that varied from 8.8 to 100%; dye and hydroalcoholic extract were the most promising inhibitors. In view of this ovicidal property, O. vulgare may be an important source of viable antiparasitic compounds for nematodiosis control in ruminants

Genetic and phenotypic analysis in horses : parasitological and body growth parameters

Orientador : Prof. Dr. Marcelo Beltrão MolentoTese (doutorado) - Universidade Federal do Paraná, Setor de Ciências Agrárias, Programa de Pós-Graduação em Ciências Veterinárias. Defesa : Curitiba, 21/02/2018Inclui referênciasResumo: Ao longo das últimas décadas, os ciatostomíneos (pequenos estrôngilos) tornaram-se os parasitas internos mais importantes em cavalos. Sendo agentes de graves infecções parasitárias e com o controle cada vez mais difícil devido à seleção de populações resistentes à maioria dos grupos de anti-helmínticos disponíveis. Portanto, são necessários estudos para avaliar as infecções parasitárias e a influência dessas doenças no desenvolvimento corporal dos animais; assim como, descobrir os padrões genéticos que podem estar relacionados à sua infeção, apresentando alta ou baixa contagem de ovos por grama de fezes (OPG). O objetivo deste estudo foi realizar análises genéticas e fenotípicas de parâmetros parasitológicos e de desenvolvimento corporal em cavalos de puro sangue inglês (PSI) naturalmente parasitados. A presente tese está dividida em cinco capítulos, apresentados em formato de Introdução, manuscritos e as Considerações Finais. O primeiro artigo comparou duas técnicas de contagem de OPG, McMaster (McM) e Mini-FLOTAC (mF), para a detecção de ovos de nematódeos gastrointestinais de bovinos e equinos, em diferentes locais. Em todos os experimentos em média os valores do desvio padrão e do coeficiente de variação foram significativamente menores para a técnica de mF. Portanto, recomenda-se o uso da técnica MiniFLOTAC, que é um método com menor variabilidade e maior precisão, especialmente para animais com baixo OPG. O segundo artigo teve como objetivo avaliar o impacto do gênero do animal, mês de nascimento, idade e número de filhos da égua, e contagem de OPG na taxa de crescimento de cavalos PSI. Este estudo também teve como objetivo correlacionar as informações sobre o desempenho dos animais nas corridas associadas aos valores OPG. O peso corporal médio dos animais ao nascer foi 56,5 kg (machos) e 51,9 kg (fêmeas), e aos 18 meses, pesavam em média 435,9 e 441,6 kg, respectivamente. Altura da cernelha média ao nascer foi de 104,36 cm (machos) e 101,19 cm (fêmeas), e aos 18 meses, a altura foi de 154,78 e 153,5 cm, respectivamente. Os dados das corridas mostraram que os animais com maior OPG apresentaram um desempenho menor quando comparados com aqueles com menor OPG, incluindo um número menor de vitórias. Concluindo que animais criados sob um conjunto bem definido de práticas de manejo podem ter poucos ou nenhum sinal de comprometimento devido as infecções parasitárias. O objetivo do artigo 3 foi determinar regiões genômicas associadas aos padrões individuais do OPG dos animais, realizando um estudo de associação genômica ampla entre equinos PSI naturalmente infectados com parasitas gastrointestinais. Neste estudo, regiões candidatas relacionadas ao desenvolvimento e ativação do sistema imune foram encontradas nos cromosomos 17, 21 e 25. Esta tese destacou várias possibilidades para investigar parasitarias gastrointestinais em cavalos, melhorando a compreensão e o controle de infecções por ciatostomíneos. Observou-se que os cavalos criados sob um conjunto bem definido de práticas de manejo podem ter poucos ou nenhum sinal de comprometimento devido a infecções parasitárias. Palavras-chave: Equino. Ciatostomíneos. Cavalo. Desenvolvimento corporal. Mini-FLOTAC. OPG. GWAS.Abstract: Over the last decades, cyathostomins (small strongyles) have become the most important internal parasites of horses. They are agents of serious parasitic infections and their control is increasingly difficult due to the selection of resistant populations to most of the anthelmintic groups available. Therefore, studies to assess the parasite infections, and the influence of these diseases on body development of animals are necessary. In the same line, it is important to discover the genetic patterns of the animals that may be related to their infection, presenting high or low fecal egg count (FEC). The aim of this study was to perform genetic and phenotypic analysis of parasitological and body development parameters in Thoroughbred horses. The present Thesis is divided in five Chapters, presented in the format of Introduction, manuscripts and Final Considerations. The first article compared two FEC techniques, namely McMaster (McM) and Mini-FLOTAC (mF), for the detection of cattle and horse gastrointestinal nematode eggs, in different locations. On average, in all experiments, the standard deviation and the coefficient of variation values were significantly lower for mF. Therefore, it is recommended the use of the Mini-FLOTAC technique, which is a method with less variability and higher accuracy, particularly for animals with low FEC. The second article had the objective to evaluate the impact of animal gender, month of birth, age of mare, number of offspring and FEC in the growth rate of Thoroughbred horses. This study also aimed to correlate the information on racing careers associated with the FEC values of the animals. The mean body weight at birth was 56.5 kg for males and 51.9 kg for females, and at 18 months’, males and females weighed on average 435.9 and 441.6 kg, respectively. The mean withers height at birth was 104.36 cm for males and 101.19 cm for females, and at 18 months, the height was 154.78 and 153.5 cm, respectively. The racing history data showed that animals with higher FEC had a poorer performance when compared to those with lower FEC, including a smaller number of victories. In conclusion, it was suggested that horses raised under a welldefined set of management practices can have little or no signs of impairment due to worm infections. The aim of the article three was to determine genomic regions associated with Strongyle FEC patterns, conducting a genomic wide association study (GWAS) among Thoroughbred equines naturally infected with gastrointestinal parasites. In this study, candidate regions related to immune system development and activation were found in ECA17, ECA 21 and ECA25. This thesis has highlighted several possibilities to further investigate the control of gastrointestinal parasite infections in horses, improving the understanding and control of cyathostomins infections. It was observed that horses raised under a well-defined set of management practices can have little or no signs of impairment due to worm infections. Key-words: Equine. Horse. Cyathostomins. Body growth. Mini-FLOTAC. EPG. GWAS

Avaliação do potencial anti-helmíntico de Origanum vulgare e Eugenia uniflora em nematódeos gastrointestinais de ruminantes

This study aimed to evaluate the in vitro effects of Origanum vulgare extracts on gastrointestinal nematode of cattle and the in vitro and in vivo action of Eugenia uniflora extracts in Haemonchus contortus as well as estimating its toxicity in VERO cells. For the in vitro analysis of different hydroalcoholic and aqueous extracts, the technique of inhibiting the hatchability of gastrointestinal nematodes eggs was applied; the Origanum vulgare and the Eugenia uniflora extracts were tested in the concentration of 0.62-80 mg/mL and 1.25-40 mg/mL, respectively. Commercial anthelmintics and distilled water were used as control treatments. The cytotoxic effect of E. uniflora extracts was measured by using MTT assay (thiazolyl blue tetrazolium bromide test) in VERO cells. The in vivo test was conducted with naturally infected sheep receiving 100 mg/kg of the hydroalcoholic extract by maceration of E. uniflora administered orally for three consecutive days; fecal collection was performed to estimate the reduction in EPG. Test results showed that the O. vulgare extracts inhibited egg hatch of cattle gastrointestinal nematodes at a percentage that varied from 8.8 to 100%. The extracts of E. uniflora also inhibited the hatchability of H. contortus eggs, with an action level ranging from 14.56 to 99.75%. The extracts have shown moderate to null cytotoxicity, ranging from 51.23 to 100% viable cells. It was not observed the reduction of the EPG for the treatment group in the in vivo test, only for the positive control group with anthelmintic. The different O. vulgare and E. uniflora extracts have proved to be effectual on the in vitro inhibition of the hatchability of nematodes eggs in ruminants, with O. vulgare dye and hydroalcoholic extract being the most promising methods for gastrointestinal nematodes control in cattle and hydroalcoholic extracts of E. uniflora for H. contortus eggs.Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPESO estudo avaliou a ação in vitro de extratos de Origanum vulgare sobre nematódeos gastrintestinais de bovinos e a ação in vitro e in vivo de extratos de Eugenia uniflora em Haemonchus contortus, além de estimar a sua toxicidade em células VERO. Para as análises in vitro dos diferentes extratos hidroalcóolicos e aquosos foi utilizada a técnica de inibição da eclodibilidade dos nematódeos gastrintestinais, os extratos de Origanum vulgare foram testados na concentração de 0,62 a 80 mg/mL e os de Eugenia uniflora de 1,25 a 40 mg/mL. Como controle foram utilizados anti-helmíntico comercial e água destilada. O efeito citotóxico dos extratos de E. uniflora foi mensurado através do ensaio de MTT (brometo tiazoli azul de tetrazólio) em células VERO. O teste in vivo foi realizado com ovinos naturalmente infectados que receberam 100 mg/Kg do extrato hidroalcóolico por maceração de E. uniflora, por via oral, durante três dias consecutivos, sendo realizada coleta de fezes para estimar a redução do OPG. Os resultados dos testes demonstraram que os extratos de O. vulgare inibiram a eclodibilidade dos nematódeos gastrintestinais de bovinos com percentual de inibição variando de 8,8 a 100% e os extratos de E. uniflora inibiram a eclodibilidade de H. contortus com redução de 14,56 a 99,75%. Nas condições testadas, os extratos apresentaram citotoxicidade moderada a nula, com 100 a 51,23% de células viáveis. No teste in vivo não foi observado redução do OPG para o grupo tratado, apenas para o grupo controle positivo com anti-helmíntico. Os diferentes extratos de O. vulgare e E. uniflora possuem efeito in vitro na inibição da eclodibilidade de nematódeos de ruminantes, sendo a tintura e o extrato hidroalcóolico de O. vulgare as formas mais promissoras para os nematódeos gastrintestinais de bovinos e os extratos hidroalcóolicos de E. uniflora para de H. contortus

The use of body growth and kinship data from 16 generations for predicting Thoroughbred performance

Thoroughbred horses have been intensely raised for their athletic potential that is correlated with morphological parameters (i.e., body weight - BW, and withers height - WH). Optimum and consistent body development is aimed, but excessive growth rates may lead to the development of orthopedic diseases. This study aimed to generate growth rate curves and prediction models of Thoroughbred horses by analyzing BW and WH data collected monthly over 16 years of 378 animals (23.6 animals/year). The animals were checked from birth to 18 months (160 colts and 181 fillies) on a farm in the south of Brazil. A prediction performance ARIMA model was developed based on the BW and WH of the foals using a maximum and minimum range of 7320 observations. BW and WH were 54,1 kg and 102,5 cm at birth and 397,8 kg and 150,6 cm at 18 months of age, respectively. No differences were found between sex at any age. Moreover, we have established a nonlinear function for the growth curve and on average, foals were expected to get 7.4 times heavier and 1.5 times taller when animals were fully grown. Males showed greater BW uniformity than females, as females had a lower minimum BW than males. Significant statistical differences (P < 0,05%) were reported for BW and WH of foals between pairs of sires highlighting the kinship (paternal) effect on the animals’ development. Seventy-two pairs of stallions showed statistical relevance for BW and 91 for WH. The ARIMA model produced a linear trend of BW and WH for the forecasted years. In conclusion, we recommend that careful sire selection and adequate health (i.e., parasite control, vaccination), and nutrition strategies must be adopted to achieve superior body growth as estimated by the predicting model (positive scenario). The present protocol shall be used in studs worldwide to monitor horse development. The spreadsheet is available on request to the corresponding authors

Presence of eggs of Toxocara spp. and hookworms in a student environment in Rio Grande do Sul, Brazil

Environmental contamination by parasite forms was studied on a university campus in the municipality of Pelotas, Rio Grande do Sul. Soil samples were analyzed using the modified Caldwell &amp; Caldwell technique to identify parasite forms. Nematode eggs were detected in 62% of the samples. Among the parasites detected, eggs of Toxocara spp. and Ancylostomatidae were the most prevalent parasites in the studied area throughout the study period. The results demonstrated that there is significant environmental contamination, thereby representing a risk of zoonotic infection for humans frequenting the study area

-866G/A and Ins/Del polymorphisms in UCP2 gene are associated with reduced short-term weight loss in patients who underwent Roux-en-Y gastric bypass

Background: Uncoupling protein 2 (UCP2) plays an important role in energy expenditure regulation. Previous studies have associated the common -866G/A (rs659366) and Ins/ Del polymorphisms in the UCP2 gene with metabolic and obesity-related phenotypes. However, it is still unclear whether these polymorphisms influence weight loss after bariatric surgery. Objectives: To investigate whether UCP2 -866G/A and Ins/Del polymorphisms are associated with weight loss outcomes after bariatric surgery. Setting: Longitudinal study in a university hospital. Methods: We retrospectively evaluated 186 patients who underwent Roux-en-Y gastric bypass (RYGB) surgery for clinical and laboratory characteristics in the preoperative period, 6, 12, and 18 months after RYGB. The -866G/A (rs659366) polymorphism was genotyped using real-time PCR, while the Ins/Del polymorphism was genotyped by direct separation of PCR products in 2.5% agarose gels. Results: Patients with the -866A/A genotype showed higher body mass index (BMI) after 6, 12, and 18 months of surgery and excess body weight after 6 and 12 months compared with G/G patients. They also showed lower excess weight loss (EWL%) after 6 and 12 months of surgery. Ins allele carriers (Ins/Ins + Ins/Del) had lower delta (Δ) BMI 12 months after surgery compared with Del/Del patients. Accordingly, patients carrying haplotypes with >=2 risk alleles of these polymorphisms had higher BMI and excess weight and lower EWL% during follow-up. Conclusion: UCP2 -866A/A genotype is associated with higher BMI and excess weight and lower EWL% during an 18-month follow-up of patients who underwent RYGB, while the Ins allele seems to be associated with lower ΔBMI 12 months after surgery. Further studies are needed to confirm the associations of the -866G/A and Ins/Del polymorphisms with weight loss after bariatric surgery